| Nitric oxide (NO) is an important, yet enigmatic signaling molecule in the CNS for chemists and neurobiologists. Understanding the pathways of NO production, release, reaction and degradation in the CNS ultimately requires the characterization of NO production within single cells due to the limited number of nitrergic cells in biochemically complex, heterogeneous tissues of the nervous system.; Endogenous NO is generated by nitric oxide synthases (NOSs), which convert arginine (Arg) and oxygen to citrulline (Cit) and NO. Citrulline is enzymatically transformed back to arginine by argininosuccinate synthetase and argininosuccinate lyase via argininosuccinate (ArgSuc). Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection provides a scheme for efficient separation and sensitive measurement of NO and NO-related compounds in complex mass limited biological samples such as single cells. In this body of work, identified molluscan neurons have been used as models for validating the proposed methods to investigate NO production within single neurons. To validate my approaches for measuring NO in cells, I sampled NO-positive B2 and the adjacent B1 neurons from the nervous system of the freshwater snail, Lymnaea stagnalis. Significant amounts of NO are detected in these neurons particularly the B2 cell. High concentrations of ArgSuc, exceeding Cit levels, are ubiquitously present in the CNS of Aplysia californica. Based on the ArgSuc and Cit concentration changes observed following incubation with NOS metabolites, we propose that the complete Arg-Cit-NO cycle may not be present in the same neuron. The implication of this finding suggests a novel biochemical pathway in which NO production requires a well-concerted coordination from multiple neurons with efficient chemical communication between them.; To resolve NO producing neurons in complex brain tissues, NO imaging methods have been developed using a novel combination of approaches with NO-sensitive fluorescent indicators. These NO indicators cross react with ascorbic acid compounds present in all cells. Here, I show that this non-specific cross-reactive species can be distinguished from NO using ratiometric comparisons of multiple NO fluorescent indicators. We use these indicators to identify NO-producing neurons in the Aplysia CNS. Punctate fluorescence is observed in buccal motor neurons. Other large neurons, such as R2 neurons and metacerebral cells, do not fluoresce within the cell; however, fluorescence is observed on the surface of these neurons which is ascribed to NO production at axon terminals. As the fluorescence observed at axon terminals is not mediated by NOS inhibitors or by NOS metabolites, non-enzymatic NO production is proposed the origin of the NO.; These methods I have developed provide sensitive, yet robust, analytical tools for the evaluation of NO and NOS metabolites with subcellular resolution. Using these approaches, the characterization of NO production and NO metabolism can be realized at the single cell level and even from subcellular microdomains. |
